Background The objective of this study was to review our experience in the development of antisense (AS) oligodeoxynucleotide (ODN) therapy for prostate cancer targeting antiapoptotic gene, clusterin.

Methods We initially summarized our data demonstrating that clusterin could be an optimal therapeutic target for prostate cancer, then presented the process of developing AS ODN therapy using several preclinical animal models. Finally, the preliminary data of the recently completed phase I clinical trial using AS clusterin ODN as well as the future prospects of this therapy are discussed.

Results Expression of clusterin was highly up-regulated after androgen withdrawal and during progression to androgen-independence, but low or absent in untreated tissues in both prostate cancer animal model systems and human clinical specimens. Introduction of the clusterin gene into human prostate cancer cells confers resistance to several therapeutic stimuli, including androgen ablation, chemotherapy and radiation. AS ODN targeting the translation initiation site of the clusterin gene markedly inhibited clusterin expression in prostate cancer cells in a dose-dependent and sequence-specific manner. Systemic treatment with AS clusterin ODN enhanced the effects of several conventional therapies through the effective induction of apoptosis in prostate cancer xenograft models. Based on these findings, a phase I clinical trial was completed using AS clusterin ODN incorporating 2′-O-(2-methoxy)ethyl-gapmer backbone (OGX-011), showing up to 90% suppression of clusterin in prostate cancer.

Conclusions The data described above identified clusterin as an antiapoptotic gene up-regulated in an adaptive cell survival manner following various cell death triggers that helps confer a phenotype resistant to therapeutic stimuli. Inhibition of clusterin expression using AS ODN technology enhances apoptosis induced by several conventional treatments, resulting in the delay of AI progression and improved survival. Clinical trials using AS ODN confirm potent suppression of clusterin expression and phase II studies will begin in early 2005.

Androgen withdrawal therapy is the only effective form of systemic therapy for men with advanced prostate cancer, resulting in symptomatic and/or objective response in more than 80% of these patients. Androgen-independent (AI) progression, however, ultimately occurs within a few years in most of these cases, and remains the main obstacle to improving survival and quality of life in this disease.1 Although a number of non-hormonal agents have been shown to have limited antitumor activity in patients with AI prostate cancer,2 two recently completed phase III trials demonstrated significant survival benefit of docetaxel-based combination regimens in such patients.3,4 However, these improved effects were modest, suggesting the need for developing a novel therapeutic strategy against advanced prostate cancer. A more rational strategy would incorporate agents, that targets adaptive changes in gene expression precipitated by several therapeutic stimuli, including androgen ablation, chemotherapy and radiation, to enhance treatment-induced apoptosis and consequently delay emergence of the AI phenotype.

Improved understanding of the specific mechanisms mediating AI progression would be necessary for establishing a new therapeutic strategy designed to inhibit the emergence of the AI phenotype before additional advantage in survival can be achieved. Although the precise mechanism of progression to androgen-independence has not been fully elucidated, intensive efforts partially clarified the complex process during AI progression involving variable combinations of clonal selection,5 androgen receptor transactivation in the absence of androgen from mutations or increased levels of coactivators,6,7 alternative growth factor pathways8–10 and adaptive up-regulation of cell survival genes.11–15 However, to date, there has not been any therapy based on such a molecular mechanism introduced into clinical practice as a useful alternative to conventional therapeutic approaches for advanced prostate cancer.

Recent investigations demonstrated that resistance to various therapeutic stimuli develops, in part, from alternations in the apoptotic machinery because of increased activity of antiapoptotic pathways by up-regulation of antiapoptotic genes.11–20 Among these genes, we have particularly focused on clusterin, also known as testosterone-repressed prostate message-2, apolipoprotein J or sulfated glucoprotein-2,21,22 that appear to play functionally important roles in the acquisition of a therapy-resistant phenotype, as a potential therapeutic target for prostate cancer. In this review, we have attempted to summarize the progress that we have recently made in the development of novel therapeutic strategy targeting clusterin gene using antisense (AS) oligodeoxynucleotide (ODN) technology.23–25

Clusterin has been shown to be associated with a wide variety of pathophysiological processes, including tissue remodeling, lipid transport, reproduction, complement regulation and apoptotic cell death.22 Clusterin expression is highly up-regulated in various normal and malignant tissues undergoing apoptosis;22,26,27 hence, clusterin has been regarded as a marker for cell death. In the prostate gland as well, the expression level of clusterin increases dramatically during castration-induced apoptosis in rat prostatic epithelial cells,28,29 androgen-dependent mouse Shionogi tumors,12,30 and several human prostate cancer xenograft models.31–33 In addition to androgen withdrawal, clusterin expression in prostate cancer cells increases following a diverse variety of apoptotic stimuli, including cytotoxic chemotherapy,34,35 radiation,36,37 oxidative stress38 and adenoviral-mediated p53 gene transfer.39 These patterns of clusterin expression induced by therapeutic stimuli were confirmed in other types of malignant tumor models.40–44

In clinical specimens of malignant tumors, such as the prostate,18,45,46 lung,44 breast,47 ovary,48 bladder,49 and renal cell carcinoma,43,50 high levels of clusterin expression are observed. Furthermore, in some types of malignancies, expression level of clusterin has been shown to be closely associated with the survival of patients.18,45,46,49,50 Consistent with preclinical xenograft models, clusterin expression in prostate cancer specimens from patients who underwent preoperative neoadjuvant hormonal therapy was significantly greater than that from those who had not received neoadjuvant therapy (Fig. 1).

Figure 1. Immunohistochemical analysis of clusterin protein expression in radical prostatectomy specimens. Low staining of clusterin is observed in specimen from patients who did not received hormonal therapy prior to surgery (A), while strong staining intensity is detected in specimen from those treated with neoadjuvant hormonal therapy (B).

Despite its original recognition as a marker for cell death, recent studies provide conflicting findings regarding the relationship between clusterin up-regulation and increased apoptotic activity.51–53 Furthermore, clusterin has been shown to bind to a wide variety of biological ligands,54–56 and to be regulated by the transcription factor, heat shock factor-1 (HSF-1),57 suggesting that clusterin functions like a heat shock protein to chaperone and stabilize conformations of proteins at time of cell stress. Indeed, clusterin is substantially more potent than other heat shock proteins at inhibiting stress-induced protein precipitation, and may function to help stressed cells cope with the increased load of unfolded proteins. As described above, accumulating data identify clusterin as a potential inhibitor of apoptosis; however, little is known about the mechanism and location of its action at the molecular and cellular levels.

Recently, several studies reported not only antiapoptotic but also proapoptotic functions of clusterin.58–65 This ambiguity between different activities of clusterin may result from the existence of two functionally divergent isoforms;58,60,61 that is, the secreted form might play a cytoprotective role, although the intracellular form could be cytotoxic and induce cell death through its nuclear localization.59,66,67 The secreted glycosilated form is a highly conserved disulfated-linked heterodimeric sulfated glycoprotein comprised of 40 and 60 kDa subunits derived from the first ATG codon of the full length clusterin,68 while the other isoform, which translocates from the cytoplasm to the nucleus following several stimuli to induce apoptosis, starts from the second ATG codon and therefore omits the endoplasmic reticulum-targeting signal. Collectively, these findings suggest that pro- and anti-apoptotic functions of clusterin may be due to the activities of different isoforms that arise through alternative splicing of the mRNA transcript.

In prostate cancer, experimental and clinical studies have demonstrated that clusterin expression is associated with AI progression and has a protective role against a wide variety of apoptotic signals.12,18,34–39,42,45,46,69–72 For example, increased expression of clusterin in prostate cancer is closely correlated to higher Gleason score45 and worse prognosis.46 To further investigate the functional significance of clusterin, the effects of clusterin overexpression were evaluated using stably transfected human androgen-dependent prostate cancer LNCaP cells with secreted form of clusterin cDNA. Clusterin-transfected LNCaP tumors were more resistant to castration, cytototoxic chemotherapy and radiation.12,34,36,37 These findings strongly suggest that clusterin is a cytoprotective gene up-regulated by apoptotic triggers and conferring resistance to conventional therapeutic modalities used in clinical practice. Considering these findings, it would be an attractive approach for advanced prostate cancer to enhance therapy-induced apoptosis by targeting the clusterin gene up-regulated following proapoptotic stimuli.

AS ODN technology can provide strategy to specifically inhibit expression of target genes playing potentially important roles in the progression of malignant tumors. AS ODN consists of chemically modified stretches of single-strand DNA, complementary to mRNA regions of a target gene, which inhibits translation by forming RNA/DNA duplexes, thereby suppressing the mRNA and protein levels of the target genes.23–25 The most commonly accepted mechanism of AS action implicates RNase H-mediated hydrolysis of the target strand mRNA of RNA/DNA duplexes,73 while RNase-independent mechanisms are also implicated, such as steric blockade of splicing, translational arrest and the prevention of 5′-capping of the mRNA which affects nuclear transport of RNA.74–77

Phosphorothioate AS ODN is a water soluble, stable agent resistant to nuclease digestion through substitution of a non-bridging phosphoryl oxygen of DNA with sulfur.78,79 We initially used this type of AS ODN targeting several kinds of functionally relevant genes, and successfully demonstrated specific inhibition of these genes, resulting in delayed malignant progression in preclinical model systems.11–13,19,20,34,35,40,80–83 However, the incorporation of phosphorothioate modification generates a chiral center and reduces binding affinity for target mRNA, and despite showing less sensitivity to nucleases than the first generation ODN, phosphorothioate AS ODN degrades in cells over time.84 To overcome these problems that would create serious limitations in a clinical setting, recent efforts have focused on backbone modification to provide a more attractive pharmacological profile than phosphorothioate ODN. Among various modifications, the 2′-O-(2′-methoxy)ethyl (2′-MOE) incorporation was identified as enhancing both binding affinity and further resisting degradation by intracellular nucleases.85 This property results in improved tissue half-life in vivo, producing a longer duration of action and allowing for a more relaxed dosing regimen.86 We also reported both in vitro and in vivo results in the human prostate cancer PC-3 model showing a significantly improved efficacy for 2′-MOE-modified AS ODN over conventional phosphorothioate AS ODN to suppress secreted form of clusterin expression and to enhance chemotherapy-induced apoptosis;87 accordingly, we subsequently used 2′-MOE-modified AS ODN targeting the clusterin gene in the following studies.

AS ODNs corresponding to the translation initiation site of murine and human clusterin genes inhibit expression levels of murine and human secreted form of clusterin, respectively, in a dose-dependent and sequence-specific manner.12,35 Adjuvant treatment of mice bearing androgen-dependent Shionogi tumors with AS ODN targeting the secreted form of clusterin gene following castration suppressed clusterin expression by more than 70% and resulted in earlier apoptotic tumor regression with a significant delay in the recurrence of AI tumors compared to that in mice treated with control ODN.12 This outcome supports the concept that targeting the cell survival gene, clusterin, which is up-regulated by androgen ablation, could enhance castration-induced apoptotic cell death and thereby prolong the interval until AI recurrence in prostate cancer.

AS clusterin ODN also enhanced the cytotoxicity of several potential chemotherapeutic agents, such as paclitaxel, docetaxel and mitoxantrone on prostate cancer cells in vitro, reducing their IC50 by 75–90%.34,35,71 Although treatment with AS clusterin ODN alone as a single agent had no effects on then growth of established prostate tumors in vivo, AS clusterin ODN synergistically enhanced tumor regression by combined use of chemotherapeutic agents in prostate cancer xenograft models.34,35 Consistent with the prostate cancer model, we recently reported that inhibition of clusterin levels synergistically chemosensitized several cancers, including renal cell carcinoma,43 bladder cancer,40,41 lung cancer44 and osteosarcoma.77 Similarly, small interfering RNA targeting clusterin gene enhanced the effects of cytotoxic chemotherapy on prostate cancer cells.72

The precise molecular mechanism mediating resistance to radiation therapy against prostate cancer remains largely unknown. Overexpression of bcl-2, one of the most potential antiapoptotic genes, in LNCaP cells appears to be more resistant to radiation-induced apoptosis.88 Clusterin-overexpressing LNCaP cells also showed a phenotype that was significantly more resistant to irradiation with lower cell death rates compared with that shown by control cells.37 Furthermore, administration of AS clusterin ODN in combination with radiation synergistically inhibited PC3 tumor growth in vivo compared with that of control ODN plus radiation.36 These findings suggested the usefulness of AS strategy targeting antiapoptotic genes to enhance therapeutic efficacy of radiation therapy.

Our recent studies identified other proapoptotic stimuli, whose effects could be enhanced by suppressing clusterin expression using AS ODN in prostate cancer cells.38,39,89 For example, treatment of mice bearing AI PC3 tumors with AS clusterin ODN and adenoviral-mediated p53 gene transfer resulted in synergistic tumor regression compared with that of either agent alone. This combined regimen also synergistically inhibited lymph node metastases after orthotopic injection of PC3 cells into the prostate of mice compared with a single agent treatment.39 Collectively, AS ODN targeting the secreted form of clusterin gene may enhance a wide variety of therapeutic stimuli against prostate cancer, and consequently inhibit tumor progression through the effective induction of apoptosis. In Table 1, an overview of preclinical studies using AS clusterin ODN is presented.

Before moving into clinical trials, to identify the most potent AS ODN sequence for suppressing clusterin expression, a series of 80 sequences for AS ODN targeting the clusterin gene were investigated. This analysis identified a 21-mer sequence designed at the translation initiation site, which have been used in all preclinical studies, as the most effective AS ODN sequence. This 21-mer AS ODN was incorporated into the MOE-gapmer backbone and synthesized for human trials as OGX-011 under a codevelopment relationship between OncoGenex Technologies (Vancouver, Canada) and ISIS Pharmaceuticals (Carlsbad, CA). In preclinical animal studies, OGX-011 showed a significantly longer in vivo tissue half-life than phosphorothioate AS ODN (5 days vs 0.5 days). Weekly administration of OGX-011 was equivalent to daily phosphorothioate AS ODN in enhancing the cytotoxic effects of chemotherapeutic agents with no additional specific side-effects.87 These outcomes strongly support the use of OGX-011 over conventional phosphorothioate AS ODN in clinical trials.

A phase I clinical trial, NCIC IND. 153, was recently completed at The University of British Columbia using OGX-011 (Table 2). This trial was performed under a unique design; that is, patients with clinically localized prostate cancer were enrolled and were administered OGX-011 prior to radical prostatectomy in order to evaluate the pharmacodynamic endpoint in each patient and subsequently determine the optimal dose level of OGX-011. The OGX-011 was given by an intravenous infusion for 2 h on days 1, 3, 5, 8, 15, 22 and 29 with radical prostatectomy performed within 7 days of the last administration of OGX-011. Relevant concentrations of OGX-011 that inhibited expression of clusterin in human prostate cancer tissues could be achieved in a dose-dependent manner, and a biologically effective dose of 640 mg was identified based on findings showing up to 90% suppression of clusterin. These findings suggest that a well tolerated dose for phase II trials was determined based on biological effectiveness rather than the traditional endpoint of phase I trial, a maximal tolerated dose which may not always be associated with targeted therapeutics. Side-effects included fever and chills in the first week of OXG-011 infusions, and transient myelosuppression and elevations of liver enzyme tests that normalized despite continued administration of OGX-011; hence, the protocol of this trial could be completed in all patients. The phase I trial illustrated that OGX-011 is well tolerated and effectively inhibits clusterin expression in human prostate cancer. Another phase I trial, NCIC IND. 154, using OGX-011 and docetaxel for patients with solid tumors, is underway (Table 2). Furthermore, Phase II studies of OGX-011 in combination with hormonal therapy and/or cytotoxic chemotherapy are planned to start in patients with prostate, breast and lung cancers in early 2005. An overview of several AS ODNs currently evaluated in clinical trials targeting malignant tumors is shown in Table 3.

Numerous genes and cellular pathways are involved in the mechanism regulating progression to androgen-independence; therefore, inactivation of a single molecular target may likely be insufficient to suppress tumor progression. Investigating whether additional antitumor effects could be achieved by blocking several antiapoptotic genes simultaneously with AS technology may help guide the design of further clinical protocols. One such strategy is to target gene family members sharing a wide range of sequence homology with a single bispecific AS ODN that can inhibit expressions of plural genes. For example, the design and preclinical testing of a bcl-2/bcl-xL bispecific AS ODN was recently reported,90,91 and the usefulness of this ODN either alone or in combination with AS clusterin ODN in a prostate cancer model was confirmed.92 This type of approach may also involve targeting other gene family members, such as insulin-like growth factor binding protein (IGFBP)93 and inhibitors of apoptosis protein (IAP).94 Moreover, a number of additional novel therapeutic strategies for prostate cancer are being developed based on the understanding of molecular mechanisms, such as signal transduction, angiogenesis, tumor invasion and metastasis.95–98 It would also be promising to achieve synergistic benefits of AS clusterin ODN-based strategy by combining such novel agents.

The findings reviewed above demonstrated that clusterin is an antiapoptotic protein up-regulated in an adaptive cell survival fashion by a wide variety of proapoptotic stimuli that confer a resistant phenotype to prostate cancer cells against several cell death triggers, including androgen ablation, cytotoxic chemotherapy and radiation. Inhibition of clusterin using AS ODN technology can enhance apoptotic cell death induced by conventional as well as molecular-targeting treatments against prostate cancer. Phase I clinical trials with OGX-011 confirm its potent activity for suppressing clusterin expression and phase II trials of OGX-011 in combination with chemotherapeutic agents therefore will start in early 2005 at The University of British Columbia. Based on the Vancouver experience, we would like to emphasize the importance of close communication and collaboration among scientists, clinicians, industry and regulatory agencies for the challenges inherent in successful translation of an integrated molecular approach for prostate cancer into clinical settings.